Systems, methods and compositions for analysis of proteoforms
Methods, reagents, kits and systems for analyzing different proteoforms of proteins of interest are provided. The provided methods, systems, etc. provide detection, characterization and quantitation of proteoforms for different biologically relevant proteins for monitoring and characterizing biological processes.
1 . A method of analyzing proteins in a first sample, comprising:
providing a population of individual protein molecules from the first sample, wherein said individual protein molecules are immobilized upon one or more solid supports and are individually addressable, and wherein the population of individual protein molecules comprises a plurality of individual molecules of a Tau protein; and
identifying a plurality of proteoforms of the Tau protein in the first sample by identifying the proteoform represented by each of the plurality of individual molecules of Tau protein on the one or more solid supports based upon identification of a presence or absence of at least 3 different modifications within each of the individual molecules of the Tau protein.
2 . The method of claim 1 , wherein the identifying step comprises identifying a presence or absence of at least 5 different modifications to each of the individual molecules of the Tau protein.
3 . The method of claim 2 , wherein at least 3 of the at least 5 different modifications of Tau protein are selected from the group of pT181, pS202, pT205, pS214, pT217, and pT231.
4 . The method of claim 2 , wherein at least 2 of the at least 5 different modifications of Tau protein are selected from the group of ON, IN, 2N, 3R and 4R isoforms of Tau protein.
5 . The method of claim 1 , wherein the providing step comprises enriching for the individual molecules of the Tau protein from the first sample prior to immobilizing the individual molecules of the Tau protein onto the one or more solid supports.
6 . The method of claim 5 , wherein the enriching step comprises:
contacting the individual molecules of Tau protein in the first sample with beads coated with antibodies that specifically bind to Tau proteins under conditions to promote binding of the antibodies to the individual molecules of Tau protein in the first sample;
washing the beads to remove unbound material;
contacting the beads with a polypeptide that mimics or duplicates a portion of Tau protein that is bound by the antibodies under conditions whereby the polypeptide competes with the Tau protein for binding to the antibodies, thereby releasing the individual molecules of Tau protein from the antibodies; and
collecting enriched Tau protein.
7 . The method of claim 5 , comprising spiking in a known amount of a standard protein into the first sample prior to the enriching step.
8 . The method of claim 1 , wherein the identifying step comprises identifying a presence or absence of at least 7 different modifications to each of the individual molecules of the Tau protein.
9 . The method of claim 1 , wherein the identifying step comprises identifying a presence or absence of at least 10 different modifications to each of the individual molecules of the Tau protein.
10 . The method of claim 1 , wherein the one or more solid supports comprises an array surface, and the population of individual protein molecules are immobilized on individually addressable locations of the array surface.
11 . The method of claim 10 , wherein the individual protein molecules are individually coupled to separate structured nucleic acid particles which are immobilized on the array surface.
12 . The method of claim 1 , wherein the first sample comprises at least 5 different proteoforms of the Tau protein.
13 . The method of claim 12 , wherein the first sample comprises at least 20 different proteoforms of the Tau protein.
14 . The method of claim 1 , wherein the identifying step is configured to identify at least 5 different proteoforms of the Tau protein.
15 . The method of claim 14 , wherein the identifying step is configured to identify at least 20 different proteoforms of the Tau protein.
16 . The method of claim 15 , wherein the identifying step is configured to identify at least 100 different proteoforms of the Tau protein.
17 . The method of claim 1 , further comprising the step of quantifying an amount of each of the plurality of different proteoforms of the Tau protein in the first sample identified in the identifying step.
18 . The method of claim 1 , wherein identifying the presence or absence of modifications within each individual molecule of the Tau protein comprises:
contacting the individual molecules of the Tau protein with a plurality of affinity reagents, wherein each of the plurality of affinity reagents comprises a specific binding affinity for a different modification to the Tau protein; and
detecting whether each of the plurality of affinity reagents binds to individual molecules of the Tau protein.
19 . The method of claim 1 , further comprising repeating the providing and identifying steps with a population of individual protein molecules from a second sample that comprises a plurality of molecules of the Tau protein, and comparing proteoforms of the Tau protein identified from the first sample to proteoforms of the Tau protein identified from the second sample.
20 . The method of claim 19 , wherein the providing and identifying steps are repeated with a population of individual protein molecules from at least 10 different samples.
21 . The method of claim 20 , wherein the providing and identifying steps are repeated with a population of individual protein molecules from at least 100 different samples.
22 . The method of claim 21 , wherein the providing and identifying steps are repeated with a population of individual protein molecules from at least 1000 different samples.
23 . The method of claim 1 , wherein the population of individual protein molecules comprises a plurality of individual protein molecules of a second protein of interest, and the identifying step further comprises identifying proteoforms of the second protein of interest.